The goals of this project are twofold: a) to develop a new technique for nanoscale Mass spectrometry imaging based on AFM based tip enhanced IR ablation (nanoIR-MS). b) to apply this technique towards the application of Single cell imaging. Information on the chemical composition within a cell has implications in the understanding of cell metabolism, division, disease states, ecological effects etc. Given current technology limitations, most current analyses of biological systems are performed on groups of cells with the assumption that an ensemble average from the group will yield a useful result. However, this typically is not a valid assumption as cells of the same type exhibit diverse metabolic makeup depending on their phase in the cycle, history and interaction with the environment. Thus it is important that cells be analyzed individually in order to detect rare cells (e.g. circulating tumor cells), transient cell states, the influence of the cell environment on cells and states and aid in the understanding of differences in gene expression, protein levels, and small- molecule distributions at the single cell level. Cell heterogeneity is particularly significant in the -omis fields such as genomics, proteomics, lipidomics, and metabolomics that characterize biological systems at a molecular level. This significance led to the NIH launching a special focus program on Single cell Analysis Tools in late 2011. The size of mammalian cells is on the order of 10 m and therefore the imaging of single cells requires imaging spatial resolution of at least 1m. The nanoIR-MS technology has a potential spatial resolution of at least 10x better than this or 100 nm which offers the possibility of the imaging of biomolecules in organelles. But to achieve an innovative and commercially successful product from this proposal, 1 m spatial resolution would suffice. One of the Specific Aims of this proposal is to demonstrate that the nanoIR-MS technique can be applied for Single Cell Imaging. We will demonstrate this on two types of Single cells: Cells from Mouse brain and also to identify single Circulating Tumor cells (CTCs). As reiterated in our Letter of Support from our collaborator, Prof. Yeh who is an Oncology research surgeon, CTCs are the fundamental entities primarily responsible for spawning metastatic disease and there is a current lack of characterization technologies to identify them . To cure epithelial-based cancers-such as cancers of the breast, prostate, lung, colon and pancreas-therapies need to be directed towards those cells that cause metastases. However, the majority of metastatic lesions are never biopsied due to anatomic inaccessibility or associated morbidity of the procedure. CTCs offer a readily accessible means of studying the biology of metastatic cells throughout the course of disease and are often referred to as 'Liquid Biopsy'.